The kidney filters and reabsorbs large amounts of calcium. The distal convoluted tubule is the primary site for hormonal regulation of this process. Relatively little is known about the mechanisms regulating calcium transport, however. A calcium-sensing receptor (CaR) is present in the distal tubule but the role of this receptor has been previously unknown. An expected role for the CaR would be to inhibit Ca2+ reabsorption during hypercalcemia, resulting in increased urinary loss of Ca2+. We have preliminary data that the CaR inhibits Ca2+ transport in a distal tubule cell culture model and that this regulation requires a protein kinase. We hypothesize that Ca2+ inhibits its own transport through decreased activity of the plasma membrane Ca2+ ATPase. This proposal will investigate the signal transduction pathways the CaR uses to regulate Ca2+ transport in MDCK and DCT cells. The specific molecules necessary for regulation of Ca2+ transport will be determined using specific pharmacological and dominant negative inhibitors of these signaling molecules. A similar approach will be used to determine which signaling pathways are necessary for Ca2+ extrusion and uptake. Activation of second messengers by the CaR will be confirmed. Finally the direct effects of extracellular calcium on phosphorylation state, surface localization and activity of the plasma membrane calcium ATPase, which pumps Ca2+ out of the cell, will be determined. The anticipated result of these studies is an understanding of the signal transduction cascade that is responsible for regulation of calcium transport. These studies will help determine the mechanisms that regulate calcium excretion and reabsorption in the distal tubule. Understanding these mechanisms will help elucidate both the normal physiology of calcium homeostasis as well as to determine more effective treatments for hypercalcemia associated with malignancy, hyperparathyroidism and other causes and with the chronic loss of calcium which occurs with osteoporosis and as a result of immunosupression.